The long-term objective of this project is to understand the functional properties of the myogenic regulatory factor (MRF) family of genes. These genes exist in animals ranging from jellyfish to humans, and are typically active only in muscle or muscle precursor cells where they play key roles in muscle development. MRF genes have also been shown to be involved in muscle repair and regeneration following injury or disuse, and to play significant roles in certain muscle abnormalities and disease states. Thus their study has clear implications for issues of human health. This project uses embryos of the ascidian, Ciona intestinalis, as a simple in vivo test system to investigate the properties of MRF genes. We plan two approaches. One is to determine whether protein motifs known to be important for vertebrate MRF activities are also important for the function of the Ciona MRF gene, Ci-MRF. We will do this by making Ci-MRF proteins with mutations in these motifs and then assaying the mutant proteins for the ability to stimulate muscle development in non-muscle cells of Ciona embryos;previously we showed that normal (i.e. non-mutant) Ci-MRF proteins stimulated muscle development in this assay. Our second approach is to test MRFs from a wide variety of other animals for the ability to direct muscle development in Ciona embryos. In these experiments, an MRF from another animal (a "heterologous" MRF) is introduced into Ci-MRF deficient Ciona embryos, which do not make muscle. We will then determine which heterologous MRFs rescue muscle development. In both approaches simple, highly specific markers of muscle development and differentiation are used to analyze experimental outcomes. Because ascidians are tunicates, the closest living relatives of the vertebrates, our studies will provide insights that are highly relevant to the functions of MRFs in vertebrate muscle development as well as insight into the fundamental properties of the MRF gene family. Public Health Relevance: The goal of this project is to understand the functions of the myogenic regulatory factor (MRF) family of genes. MRF genes play key roles in muscle development, in muscle repair, and regeneration following injury or disuse, and in certain muscle abnormalities and disease states. Thus, understanding the specific functions of MRF genes has clear relevance for human health.